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(A) Secondary structure of TetR obtained from protein data bank (PBD) with focus on the E71 and G72 (highlighted by red box) that form a turn between α-helix-4 and 5. Mutating both amino acids may cause a conformational change in rtTA. (B) Annotation of TetR protein sequence and position of the ten α-helices.
Source publication
Regulated expression of genetic elements that either encode polypeptides or various types of functional RNA is a fundamental goal for gene therapy. Inducible expression may be preferred over constitutive promoters to allow clinician-based control of gene expression. Existing Tet-On systems represent one of the tightest rheostats for control of gene...
Context in source publication
Context 1
... as compared to the original TetR: E71K, D95N, L101S and G102D 2 . In TetR, E71 is a surface residue amino acid, D95 connects the DNA reading head to the core domain, while L101 and G102 are crucial for dimerisation and the tetracycline response, respectively 2 . In TetR the E71 and G72 amino acids create the turn between α-helix-4 and 5 (Fig. 6). This region bridges the DNA binding domain to the tetracycline binding domain and the combination of both the E71K and G72V mutations might destroy the structure of this critical turn, causing a loss of rtTA-M2 activity. This may also explain the drop in tetracycline-induction observed with the position 320 mutant (E107 to Q107, Fig. ...
Citations
... Like many other gene-inducible systems, the Tet-ON system might also suffer from background "leaky" expression in the absence of an inducer, potentially limiting widespread use for clinical applications. However, researchers continuously work on further improvement of the Tet-ON system's tightness and foldresponse to make it more suitable for CAR T cell therapy [96]. ...
Cancer immunotherapies utilizing genetically engineered T cells have emerged as powerful personalized therapeutic agents showing dramatic preclinical and clinical results, particularly in hematological malignancies. Ectopically expressed chimeric antigen receptors (CARs) reprogram immune cells to target and eliminate cancer. However, CAR T cell therapy's success depends on the balance between effective anti-tumor activity and minimizing harmful side effects. To improve CAR T cell therapy outcomes and mitigate associated toxicities, scientists from different fields are cooperating in developing next-generation products using the latest molecular cell biology and synthetic biology tools and technologies. The immunotherapy field is rapidly evolving, with new approaches and strategies being reported at a fast pace. This comprehensive literature review aims to provide an up-to-date overview of the latest developments in controlling CAR T cell activity for improved safety, efficacy, and flexibility.
... Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cancers14194854/s1, Figure S1: Tet-On induction of c-FLIP protects Jurkat T cells against cell-death induced by CD95L challenge; Figure S2: Tet-On induction of c-FLIP protects Jurkat T cells against mitochondrial depolarisation mediated by CD95L challenge; Figure S3: Perforin and granzyme B production of c-FLIP-expressing CAR T cells [69][70][71][72][73][74]. Informed Consent Statement: Informed consent was obtained from all subjects involved in this study. ...
CAR T cell treatment of solid tumours is limited by poor persistence due to CD95 ligand (CD95L)-induced apoptosis. Both T cells and cells within the tumour microenvironment (TME) may express high levels of CD95L, triggering apoptosis in CD95-receptor-positive CAR T cells. Tonic signalling of CAR T cells may also increase CD95-dependent AICD. Because the intracellular protein c-FLIP protects T cells from AICD, we expressed c-FLIPp43 within a Her-2 targeted CAR cassette and evaluated the potential of c-FLIPp43 through in vitro functional assays and in vivo tumour-bearing xenograft model. cFLIP expression protected against CD95L-induced cell death in the Jurkat T cell lines. However, in primary human CAR T cells containing CAR-CD28 domains, c-FLIPp43 overexpression had minimal additional impact on resistance to CD95L-induded cell death. In vitro cytotoxicity against a breast cancer tumour cell line was not altered by c-FLIPp43 expression, but the expression of c-FLIPp43 in Her2-CAR T cells lowered interferon-γ secretion, without markedly affecting IL-2 levels, and c-FLIPp43-Her2-CAR T cells showed reduced anti-tumour activity in immunodeficient mice with breast cancer. The findings of this study provide a new understanding of the effects of controlling extrinsic apoptosis pathway suppression in CAR T cells, suggesting that c-FLIPp43 expression reduces anti-tumour immunity through the modulation of effector T cell pathways.
... We recently utilised a Sleeping Beauty (SB)-based system for expression of long and complex multi-gene chimeric antigen receptor (CAR) based cassette [7]. This system contained multiple genetic elements, including a reporter, an anti-Her2 CAR, together with an additional gene of interest (GOI: e.g. ...
... We noted significant basal expression and loss of tetracycline-mediated fold-induction in this multigene system. However, more marked improvements were noted by removal of cryptic splice sites with the most profound improvement introduced by a G72V mutation in rtTA-M2 [7,8]. Our earlier study confirmed the suitability of the G72V rtTA mutation, first described in yeast gene-regulation studies [8], for use in mammalian systems. ...
... Our earlier study confirmed the suitability of the G72V rtTA mutation, first described in yeast gene-regulation studies [8], for use in mammalian systems. We also confirmed the suitability of minimal promoter elements for tight expression in a Sleeping Beauty transposon cassette but obtained additional improvements through alterations in gene orientation and removal of cryptic splice sites in the rtTA [7]. ...
Tetracycline-inducible systems are widely used control elements for mammalian gene expression. Despite multiple iterations to improve inducibility, their use is still compromised by basal promoter activity in the absence of tetracyclines. In a mammalian system, we previously showed that the introduction of the G72V mutation in the rtTA-M2 tetracycline activator lowers the basal level expression and increases the fold-induction of multiple genetic elements in a long chimeric antigen receptor construct. In this study, we confirmed that the G72V mutation was effective in minimising background expression in the absence of an inducer, resulting in an increase in fold-expression. Loss of responsiveness due to the G72V mutation was compensated through the incorporation of four sensitivity enhancing (SE) mutations, without compromising promoter tightness. However, SE mutations alone (without G72V) led to undesirable leakiness. Although cryptic splice site removal from rtTA did not alter the inducible control of the luciferase reporter gene in this simplified vector system, this is still recommended as a precaution in more complex multi-gene elements that contain rtTA. The optimized expression construct containing G72V and SE mutations currently provides the best improvement of fold-induction mediated by the rtTA-M2 activator in a mammalian system.
... 42 Therefore, the Tet-on system has been applied to develop a tissuespecific delivery system on cell therapy and generate controllable transgene expression systems to produce animal models of disease. [43][44][45] Herein, we first generated inducible cell lines to quantify It has been shown recently that α-helix-mediated protein-protein interactions are promising drug targets that can be affected by various types of inhibitors including peptides, foldamers, and proteomimetic-derived ligands. 46 To verify the specificity of the inducible system, we have tested the inhibitory effect of potential peptide inhibitors of ZIKV C-C interaction. ...
The global spread of the mosquito‐borne Zika virus (ZIKV) infection and its complications including Guillain‐Barré syndrome and fetus microcephaly in 2015 have made ZIKV as a significant public health threat. The capsid protein plays crucial roles in ZIKV replication and thus represents an attractive therapeutic target. However, inhibitors of ZIKV capsid assembly have not been rigorously identified due to the lack of a target‐based screening system. In this study, we developed a novel ZIKV capsid interaction method based on a split‐luciferase complementation assay (SLCA), which can be used to measure and quantify ZIKV capsid‐capsid interaction by the restored luciferase signal when capsid proteins interact with each other. Furthermore, a Tet‐on inducible stable cell line was generated to screen inhibitors of capsid dimerization. By using of this system, peptides (Pep.15‐24 in the N‐terminal region of ZIKV capsid protein and Pep.44‐58 in the α2 helix of ZIKV capsid protein) were identified to inhibit ZIKV capsid‐capsid interaction. Overall, this study developed a novel inducible assay system to measure ZIKV capsid interaction and identify ZIKV capsid multimerization inhibitors, which will be applied for future discovery of ZIKV assembly inhibitors. This article is protected by copyright. All rights reserved.
... A solution to the basal activity of the Tet-on system has also been proposed in the CAR context, by introducing the G72V mutation in the TetR region. This resulted in a 40-fold reduction in Dox-independent activation [54]. An interesting study that proposed the possibility of additional control coupled the activation of the Tet system, not only to Tc but also to blue light similar to the Cre system [55]. ...
Among the many oncology therapies, few have generated as much excitement as CAR-T. The success of CAR therapy would not have been possible without the many discoveries that preceded it, most notably, the Nobel Prize-winning breakthroughs in cellular immunity. However, despite the fact that CAR-T already offers not only hope for development, but measurable results in the treatment of hematological malignancies, CAR-T still cannot be safely applied to solid tumors. The reason for this is, among other things, the lack of tumor-specific antigens which, in therapy, threatens to cause a lethal attack of lymphocytes on healthy cells. In the case of hematological malignancies, dangerous complications such as cytokine release syndrome may occur. Scientists have responded to these clinical challenges with molecular switches. They make it possible to remotely control CAR lymphocytes after they have already been administered to the patient. Moreover, they offer many additional capabilities. For example, they can be used to switch CAR antigenic specificity, create logic gates, or produce local activation under heat or light. They can also be coupled with costimulatory domains, used for the regulation of interleukin secretion, or to prevent CAR exhaustion. More complex modifications will probably require a combination of reprogramming (iPSc) technology with genome editing (CRISPR) and allogenic (off the shelf) CAR-T production.
... We showed that introducing a G72V mutation in rtTA (G72V-rtTA) described previously for yeast, 105 significantly enhanced the Tet-On system function in large gene cassettes containing a CAR. 106 Such a system might be beneficial when the expression of a miRNA needs to be regulated during T cell differentiation. For instance, let-7 has an increased expression within T N and T M cells whilst is downregulated in T EFF cells. ...
Advances made in chimeric antigen receptor (CAR) T cell therapy have revolutionized the treatment and management of certain cancers. Currently, B cell malignancies have been among the few cancers to which CAR T cells have shown persistent and resilient anti‐tumor responses. A growing body of evidence suggests that the persistence of CAR T cells within patients following infusion is linked to the mitochondrial fitness of the CAR T cell, which could affect clinical outcomes. Analysis of CAR T cells from patients undergoing successful treatment has shown an increase in mitochondrial mass and fusion events, and a reduction in aerobic metabolism, highlighting the importance of mitochondria in CAR T cell function. Consequently, there has been recent interest and investment in approaches that focus on mitochondrial programming. In this regard, miRNAs are promising agents in mitochondrial reprogramming for several reasons: (1) natural and artificial miRNAs are non‐immunogenic, (2) one miRNA can simultaneously modulate the expression of multiple genes within a pathway, (3) the small size of a sequence required for producing mature miRNA is ideal for use in viral vectors and (4) different precursor miRNAs (pre‐miRNAs) hairpins can be incorporated into a polycistronic miRNA cluster to create a miRNA cocktail. In this perspective, we describe the latest genetic engineering strategies that can be used to achieve the optimal expression of candidate miRNAs alongside a CAR construct. In addition, we include an in silico analysis of rational candidate miRNAs that could promote the mitochondrial fitness of CAR T cells. This article discusses the potential use of microRNAs to reprogram chimeric antigen receptor (CAR) T cell metabolism. We provide rationally selected target genes, the miRNAs regulating them, and the criteria for selecting miRNAs. We also describe the genetic constructs to overexpress miRNAs within CAR in T cells.
... The constitutive expression can be achieved using dual-promoter systems [132], bidirectional promoters [133], 2A self-cleaving peptides [52] or adding internal ribosome entry site (IRES) sequences [134]. For controlled expression over gene-of-interest (GOI), tetracycline (Tet)-On/Off system [135] or endogenous inducible promoters (e.g., IL-2 minimal promoter) [136] can be employed. The use of base-editing using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based technologies can introduce mutation(s) in the coding region of DNA, resulting in mutant enzymes that no longer bind to their natural ligand allowing for more precise control of genes with minimal side effects [137]. ...
... Overall, whilst there is great promise in incorporating gene manipulation in CAR T cell therapy there are also disadvantages that limit the feasibility of the approach. Firstly, although several tools have been used to control GOI expression, the basal expression or immunogenicity of transactivators in such systems restrain their performance [135,141]. Secondly, the expression patterns of genes within T cells are still largely unknown, and uncertainty surrounding the extent of the interactions between transgenes and their endogenous regulators. Continuous expression or inhibition of a gene may affect other pathways with unpredicted outcomes. ...
Simple Summary
We review the mechanisms of cellular metabolism and mitochondrial function that have potential to impact on the success of chimeric antigen receptor (CAR) T cell therapy. The review focuses readers on mitochondrial functions to allow a better understanding of the complexity of T cell metabolic pathways, energetics and apoptotic/antiapoptotic pathways occurring in CAR T cells. We highlight potential modifications of T cell metabolism and mitochondrial function for the benefit of improved adoptive cellular therapy. Reprogramming metabolism in CAR T cells is an attractive approach to improve antitumour functions, increase persistence and enable adaptation to the nutrient-restricted solid tumour environment.
Abstract
Chimeric antigen receptor (CAR) T-cell therapy has revolutionized adoptive cell therapy with impressive therapeutic outcomes of >80% complete remission (CR) rates in some haematological malignancies. Despite this, CAR T cell therapy for the treatment of solid tumours has invariably been unsuccessful in the clinic. Immunosuppressive factors and metabolic stresses in the tumour microenvironment (TME) result in the dysfunction and exhaustion of CAR T cells. A growing body of evidence demonstrates the importance of the mitochondrial and metabolic state of CAR T cells prior to infusion into patients. The different T cell subtypes utilise distinct metabolic pathways to fulfil their energy demands associated with their function. The reprogramming of CAR T cell metabolism is a viable approach to manufacture CAR T cells with superior antitumour functions and increased longevity, whilst also facilitating their adaptation to the nutrient restricted TME. This review discusses the mitochondrial and metabolic state of T cells, and describes the potential of the latest metabolic interventions to maximise CAR T cell efficacy for solid tumours.
... We investigated the utility of bidirectional 2 of 9 transgene expression for driving stable gene expression within a genome-integrated Sleeping Beauty system. While developing a Tet-On Sleeping Beauty system for another study [12], we noted strong interference from an RPBSA promoter placed divergently and directly upstream of the Tet-On promoter. Removal of the RPBSA promoter negated this retrograde interference (unpublished data). ...
Promoter choice is an essential consideration for transgene expression in gene therapy. The expression of multiple genes requires ribosomal entry or skip sites, or the use of multiple promoters. Promoter systems comprised of two separate, divergent promoters may significantly increase the size of genetic cassettes intended for use in gene therapy. However, an alternative approach is to use a single, compact, bidirectional promoter. We identified strong and stable bidirectional activity of the RPBSA synthetic promoter comprised of a fragment of the human Rpl13a promoter, together with additional intron/exon structures. The Rpl13a-based promoter drove long-term bidirectional activity of fluorescent proteins. Similar results were obtained for the EF1-α and LMP2/TAP1 promoters. However, in a lentiviral vector, the divergent bidirectional systems failed to produce sufficient titres to translate into an expression system for dual chimeric antigen receptor (CAR) expression. Although bidirectional promoters show excellent applicability to drive short RNA in Sleeping Beauty transposon systems, their possible use in the lentiviral applications requiring longer and more complex RNA, such as dual-CAR cassettes, is limited.
The fast‐growing interest in cell and gene therapy (C>) products has led to a growing demand for the production of plasmid DNA (pDNA) and viral vectors for clinical and commercial use. Manufacturers, regulators, and suppliers need to develop strategies for establishing robust and agile supply chains in the otherwise empirical field of C>. In this article, we present a model‐based methodology that has great potential to support the wider adoption of C>, by ensuring efficient timelines, scalability, and cost‐effectiveness in the production of key raw materials. Specifically, we identify key process and economic parameters for (1) the production of pDNA for the forward‐looking scenario of non‐viral‐based Chimeric Antigen Receptor (CAR) T‐cell therapies from clinical (200 doses) to commercial (40,000 doses) scale and (2) the commercial (40,000 doses) production of pDNA and lentiviral vectors for the current state‐of‐the‐art viral vector‐based CAR T‐cell therapies. By applying a systematic global sensitivity analysis, we quantify uncertainty in the manufacturing process and apportion it to key process and economic parameters, highlighting cost drivers and limitations that steer decision‐making. The results underline the cost‐efficiency and operational flexibility of non‐viral‐based therapies in the overall C> supply chain, as well as the importance of economies‐of‐scale in the production of pDNA.
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CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrations have been developed, but most strategies either generate unwanted genomic scars or rely on permanent integration and expression of a reporter gene used for selection. However, stable expression of a reporter gene may perturb cell homeostasis and function. Here we develop a broadly applicable and versatile enrichment strategy by harnessing the capability of CRISPR activation (CRISPRa) to transiently induce expression of a therapeutically relevant reporter gene used for immunomagnetic enrichment. This strategy is readily adaptable to primary human T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs), where enrichment of 1.8- to 3.3-fold and 3.2- to 3.6-fold was achieved, respectively. Furthermore, chimeric antigen receptor (CAR) T cells were enriched 2.5-fold and demonstrated improved cytotoxicity over non-enriched CAR T cells. Analysis of HDR integrations showed a proportion of cells harboring deletions of the transgene cassette arising either from impartial HDR or truncated adeno-associated virus (AAV) vector genomes. Nonetheless, this novel enrichment strategy expands the possibility to enrich for transgene integrations in research settings and in gene and cellular therapies.
